An assay apparatus utilizing attenuated total reflection has been known as a device for assaying reactions of samples, for example, for the purpose of studying interaction of protein, DNA and various biochemical materials, or screening drugs.
A surface plasmon resonance (SPR) sensor is known as such an assay apparatus that utilizes attenuated total reflection. Surface plasmon is a term to mean a compressional wave of free electrons, which moves on the surface of a metal and is caused by mass oscillation of the free electrons inside the metal.
For example, Japanese Patent No. 3294605 discloses an SPR assay apparatus which adopts Kretschmann configuration, wherein a sensor surface is provided as an obverse surface of a meta film formed on a transparent dielectric material, hereinafter called a prism. A sample is reacted on this sensor surface, and thereafter, the metal film is illuminated from the back through the prism in a manner conditioned for total reflection, to measure the reflected light.
Although the light projected onto the metal film is conditioned for total reflection, a small component of the light is not reflected but passes through the metal film and penetrates to the sensor surface. The penetrating light component is called an evanescent wave. When the frequency of the evanescent wave coincides with that of the surface plasmon, surface plasmon resonance (SPR) occurs, which attenuates the intensity of the reflected light remarkably. The incident angle of the attenuating light component, that is called resonance angle, varies depending upon the refraction index on the metal film. So the SPR assay apparatus detects the resonance angle of the reflected light from the metal film, to measure the degree of reaction of the sample on the sensor surface.
For the purpose of preventing modification or deactivation due to drying, the biological sample, such as protein or DNA, is often dissolved in a solvent, to be handled as a sample fluid, wherein examples of the solvents include physiological saline water, pure water, liquid buffers and the like. The above-mentioned prior art discloses the assay apparatus for analyzing interaction of the sample, and the assay apparatus has a flow channel for flowing a sample fluid on a sensor surface. The flow channel and the prism are placed on an assay stage that is built in the apparatus. Specifically, a sensor unit chip consisting of a metal film formed on a glass support is positioned on the assay stage for the above-described measuring step.
According to the above prior art, the sample fluid is fed from a fluid container to the flow channel through a tube that is connected to a pump and valves. There is a problem in this configuration that the sample can adhere to the interior of the tube and mix with another sample fluid that flows through the tube afterward. Such a problem is called contamination.
To solve this problem, an SPR assay apparatus has been suggested, which uses pipettes for feeding a fluid, such as a sample fluid, from a container to a flow channel. The pipette consists of a pipette tip and a pipette head holding the pipette tip removably. The pipette tip is of a substantially conical-tube shape, and has a small hole at its end. In this SPR assay apparatus, each time a different fluid is to be fed, the pipette tip is changed to prevent the contamination of the fluid as being fed to the flow channel.
Besides using the pipettes, the just-mentioned assay apparatus uses a sensor unit that consists of a flow channel block having a flow channel formed therein, a prism having a metal film formed on its top surface, and a holding member. The holding member holds the flow channel member and the prism such that a bottom surface of the flow channel member is put on the top surface of the prism, i.e., the flow channel is opposed to the metal film. The flow channel is a substantially U-shaped tube formed by hollowing out the flow channel member. Opposite ends of the flow channel are exposed on the top surface of the flow channel member. To feed the fluid into the flow channel, the pipette tip is inserted in the ends of the flow channel, and the fluid is dispensed from the pipette. The flow channel member is formed from a soft elastic material, so that the flow channel member is resiliently deformed into tight contact with the prism, when they are pressed together by the holding member. Thereby, the flow channel gets highly water-tight.
However, the flow channel formed from the elastic material has a problem that the flow channel can be deformed by the inserted pipette tip or the depth of insertion of the pipette tip can vary, which results in changing the volume of the flow channel. If the volume of the flow channel varies, the flow rate of the fluid varies, so the fluid can overflow or different kinds of fluids can mix in the flow channel. The movement of the pipette into and out of the flow channel vibrates the fluid to wave in the flow channel, and can cause a measurement error if the fluid waves before the measurement. Besides that, the pipette tip can fit in the flow channel so tightly that it comes off the pipette head as the pipette is pulled out from the flow channel. Moreover, the repetitive insertion and removal of the pipette can decay the flow channel member and cause clogging of the flow channel.
Thus, the insertion of the pipette in the flow channel causes various troubles. When the pipette tip accidentally separates from the pipette head or the flow channel is clogged, the operation of the assay apparatus should be interrupted in order to deal with the trouble. Since the volume variation of the flow channel can cause measurement errors, the above-described assaying method is not sufficiently stable in view of the performance and the accuracy as well.